Chromatin is the physiological form of eukaryotic genomes and is composed of DNA and its intimately associated proteins. Histones comprise the major protein component of chromatin, and histone variant proteins are currently at the 'center stage' of histone biology due to their ability to alter the structure and function of the nucleosome. This proposal focuses on the H2A family of variants, in particular, macroH2A isoforms, which are associated with certain forms of condensed chromatin and fine-tuning of gene expression programs during development. We propose that disruption of chromatin structure can contribute to disease initiation and/or progression. In particular, we are focused on malignant melanoma due to its rising incidence, high rate of mortality and lack of effective non-surgical treatments. Our work has identified a global loss of macroH2A isoforms in malignant melanoma, both in cells and cohorts of human tissue samples. Furthermore, we have uncovered a novel role for macroH2A as a suppressor of melanoma progression. Through transcriptional profiling studies, we have identified genes regulated by this unique histone variant, focusing our studies on CDK8. CDK8 is a bone fide macroH2A target gene and contributes to cellular proliferation of murine and human melanoma cells (manuscript in press, Nature). We hypothesize that loss of macroH2A isoforms during melanoma progression significantly affects both gene expression programs and chromatin structure, contributing to progression of this disease. Therefore, deciphering the role of this histone variant will facilitate a deeper understanding of melanoma progression, and potentially lead to the identification of novel gene targets for melanoma therapies. Three aims are proposed to test these hypotheses. The first aim is designed to test the contribution(s) of CDK8 to migration and metastasis, and to determine whether the kinase activity of CDK8 is required for these processes and/or proliferation in human melanoma cells. Aim 2 is designed to validate and functionally characterize human macroH2A-regulated gene targets in melanoma using in vitro and in vivo systems. The third aim explores the downstream consequences of macroH2A loss on the chromatin template and utilizes a ChIP-high throughput sequencing approach to map the genome-wide occupancy of macroH2A in melanoma. In summary, we aim to make a significant impact in this newly emerging area of chromatin biology as it applies to melanoma biology, and identify novel and innovative strategies for potential epigenetic therapies of melanoma.